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Posterior capsule opacification (PCO) is the most common complication of cataract surgery, and intraocular lens (IOL) implantation is the standard of care for cataract patients. Induction of post-operative epithelial-mesenchymal transition (EMT) in residual lens epithelial cells (LEC) is the main mechanism by which PCO forms. Previous studies have shown that IOLs made with different materials have varying incidence of PCO. The aim of this paper was to study the interactions between human (h)LEC and polymer substrates. Polymers and copolymers of 2-hydroxyethyl methacrylate (HEMA) and 3-methacryloxypropyl tris (trimethylsiloxy) silane (TRIS) were synthesized and evaluated due to the clinical use of these materials as ocular biomaterials and implants. The chemical properties of the polymer surfaces were evaluated by contact angle, and polymer stiffness and roughness were measured using atomic force microscopy. In vitro studies showed the effect of polymer mechanical properties on the behavior of hLECs. Stiffer polymers increased α-smooth muscle actin expression and induced cell elongation. Hydrophobic and rough polymer surfaces increased cell attachment. These results demonstrate that attachment of hLECs on different surfaces is affected by surface properties in vitro, and evaluating these properties may be useful for investigating prevention of PCO.
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The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a powerful solution to rapidly advance the study of matrix- and surface-bound EVs and other particles. The versatility of LEVA is demonstrated using commercial GFP-EV standards, EVs from glioblastoma bioreactors, and E. coli outer membrane vesicles (OMVs), with the resulting patterns used for single EV characterization, single cell migration on migrasome-mimetic trails, and OMV-mediated neutrophil swarming. LEVA will enable rapid advancements in the study of matrix- and surface-bound EVs and other particles, and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.
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Regenerative medicine in tissue engineering often relies on stem cells and specific growth factors at a supraphysiological dose. These approaches are costly and may cause severe side effects. Herein, therapeutic small extracellular vesicles (t-sEVs) endogenously loaded with a cocktail of human vascular endothelial growth factor A (VEGF-A) and human bone morphogenetic protein 2 (BMP-2) mRNAs within a customized injectable PEGylated poly (glycerol sebacate) acrylate (PEGS-A) hydrogel for bone regeneration in rats with challenging femur critical-size defects are introduced. Abundant t-sEVs are produced by a facile cellular nanoelectroporation system based on a commercially available track-etched membrane (TM-nanoEP) to deliver plasmid DNAs to human adipose-derived mesenchymal stem cells (hAdMSCs). Upregulated microRNAs associated with the therapeutic mRNAs are enriched in t-sEVs for enhanced angiogenic-osteogenic regeneration. Localized and controlled release of t-sEVs within the PEGS-A hydrogel leads to the retention of therapeutics in the defect site for highly efficient bone regeneration with minimal low accumulation in other organs.
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Osteogênese , Fator A de Crescimento do Endotélio Vascular , Ratos , Humanos , Animais , RNA Mensageiro/genética , Regeneração Óssea/genética , Hidrogéis/farmacologiaRESUMO
Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.
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Saccharomycetales , Polaridade Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Longevidade , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismoRESUMO
Extracellular vesicles (EVs) are micro and nanoscale packages that circulate in all bodily fluids and play an important role in intercellular communication by shuttling biomolecules to nearby and distant cells. However, producing sufficient amounts of EVs for many types of in vitro studies using standard culture methods can be challenging, and despite the success of some bioreactors in increasing EV-production, it is still largely unknown how individual culture conditions can alter the production and content of EVs. In this study, we demonstrate a simple and inexpensive micropatterning technique that can be used to produce polystyrene microtracks over a 100 mm diameter growth surface area. We then demonstrate that these microtracks can play a significant role in increasing EV production using a triple-negative breast cancer cell line (MDA-MB-231) and that these changes in EV production correlate with increases in cellular aspect ratio, alignment of the cells' long axes to the microtracks, and single-cell migration rates. These findings have implications in both biomanufacturing of EVs and potentially in enhancing the biomimicry of EVs produced in vitro.
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Vesículas Extracelulares , Reatores Biológicos , Linhagem Celular , Movimento CelularRESUMO
Highly migratory cancer cells often lead to metastasis and recurrence and are responsible for the high mortality rates in many cancers despite aggressive treatment. Recently, the migratory behavior of patient-derived glioblastoma multiforme cells on microtracks has shown potential in predicting the likelihood of recurrence, while at the same time, antimetastasis drugs have been developed which require simple yet relevant high-throughput screening systems. However, robust in vitro platforms which can reliably seed single cells and measure their migration while mimicking the physiological tumor microenvironment have not been demonstrated. In this study, we demonstrate a microfluidic device which hydrodynamically seeds single cancer cells onto stamped or femtosecond laser ablated polystyrene microtracks, promoting 1D migratory behavior due to the cells' tendency to follow topographical cues. Using time-lapse microscopy, we found that single U87 glioblastoma multiforme cells migrated more slowly on laser ablated microtracks compared to stamped microtracks of equal width and spacing (p < 0.05) and exhibited greater directional persistence on both 1D patterns compared to flat polystyrene (p < 0.05). Single-cell morphologies also differed significantly between flat and 1D patterns, with cells on 1D substrates exhibiting higher aspect ratios and less circularity (p < 0.05). This microfluidic platform could lead to automated quantification of single-cell migratory behavior due to the high predictability of hydrodynamic seeding and guided 1D migration, an important step to realizing the potential of microfluidic migration assays for drug screening and individualized medicine.
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Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. SIGNIFICANCE: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.
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Biomarcadores Tumorais/metabolismo , Separação Celular/métodos , Cistadenocarcinoma Seroso/patologia , Exossomos/metabolismo , Microfluídica/métodos , Neoplasias Ovarianas/patologia , Estudos de Casos e Controles , Cistadenocarcinoma Seroso/metabolismo , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-6/metabolismo , Neoplasias Ovarianas/metabolismo , Proteoma/análise , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.
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Fracionamento Celular/instrumentação , Exossomos/patologia , Dispositivos Lab-On-A-Chip , Neoplasias Ovarianas/patologia , Desenho de Equipamento , Feminino , HumanosRESUMO
Quantum confined materials have been extensively studied for photoluminescent applications. Due to intrinsic limitations of low biocompatibility and challenging modulation, the utilization of conventional inorganic quantum confined photoluminescent materials in bio-imaging and bio-machine interface faces critical restrictions. Here, we present aromatic cyclo-dipeptides that dimerize into quantum dots, which serve as building blocks to further self-assemble into quantum confined supramolecular structures with diverse morphologies and photoluminescence properties. Especially, the emission can be tuned from the visible region to the near-infrared region (420 nm to 820 nm) by modulating the self-assembly process. Moreover, no obvious cytotoxic effect is observed for these nanostructures, and their utilization for in vivo imaging and as phosphors for light-emitting diodes is demonstrated. The data reveal that the morphologies and optical properties of the aromatic cyclo-dipeptide self-assemblies can be tuned, making them potential candidates for supramolecular quantum confined materials providing biocompatible alternatives for broad biomedical and opto-electric applications.
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Peptídeos/química , Pontos Quânticos/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular , Dimerização , Fluorescência , Humanos , Masculino , Camundongos Nus , Peptídeos Cíclicos/química , Pontos Quânticos/ultraestruturaRESUMO
INTRODUCTION: Microfabrication offers opportunities to study surface concepts focused to reduce bacterial adhesion on implants using human minimally invasive rapid screening (hMIRS). Wide information is available about cell/biomaterial interactions using eukaryotic and prokaryotic cells on surfaces of dental materials with different topographies, but studies using human being are still limited. OBJECTIVE: To evaluate a synergy of microfabrication and hMIRS to study the bacterial adhesion on micropatterned surfaces for dental materials. MATERIALS AND METHODS: Micropatterned and flat surfaces on biomedical PDMS disks were produced by soft lithography. The hMIRS approach was used to evaluate the total oral bacterial adhesion on PDMS surfaces placed in the oral cavity of five volunteers (the study was approved by the University Ethical Committee). After 24 h, the disks were analyzed using MTT assay and light microscopy. RESULTS: In the present pilot study, microwell structures were microfabricated on the PDMS surface via soft lithography with a spacing of 5 µm. Overall, bacterial adhesion did not significantly differ between the flat and micropatterned surfaces. However, individual analysis of two subjects showed greater bacterial adhesion on the micropatterned surfaces than on the flat surfaces. SIGNIFICANCE: Microfabrication and hMIRS might be implemented to study the cell/biomaterial interactions for dental materials.
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Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to â¼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.
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Neoplasias Encefálicas/patologia , Movimento Celular , Glioblastoma/patologia , Células-Tronco Neoplásicas/citologia , Animais , Apoptose , Humanos , Camundongos , Células Tumorais CultivadasRESUMO
A primary goal in bone tissue engineering is the design of implants that induce controlled, guided, and rapid healing. The events that normally lead to the integration of an implant into bone and determine the performance of the device occur mainly at the tissue-implant interface. Topographical surface modification of a biomaterial might be an efficient tool for inducing stem cell osteogenic differentiation and replace the use of biochemical stimuli. The main goal of this work was to develop micropatterned bioactive silica thin films to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) only through topographical stimuli. Line and pillar micropatterns were developed by a combination of sol-gel/soft lithography and characterized by scanning electron microscopy, atomic force microscopy, and contact angle measurements. hMSCs were cultured onto the microfabricated thin films and flat control for up to 21 days under basal conditions. The micropatterned groups induced levels of osteogenic differentiation and expression of osteoblast-associated markers higher than those of the flat controls. Via comparison of the micropatterns, the pillars caused a stronger response of the osteogenic differentiation of hMSCs with a higher level of expression of osteoblast-associated markers, ALP activity, and extracellular matrix mineralization after the cells had been cultured for 21 days. These findings suggest that specific microtopographic cues can direct hMSCs toward osteogenic differentiation.
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Células-Tronco Mesenquimais/citologia , Osteoclastos/citologia , Dióxido de Silício/química , Diferenciação Celular , Células Cultivadas , Humanos , Microtecnologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia TecidualRESUMO
Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation.
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Adesões Focais/fisiologia , Mucosa Respiratória/lesões , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Adesão Celular , Células Cultivadas , Humanos , Complacência Pulmonar , Mucosa Respiratória/citologiaRESUMO
Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.
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Electrically conducting polymers (CPs) were found to stimulate various cell types such as neurons, osteoblasts, and fibroblasts in both in vitro and in vivo studies. However, to our knowledge, no studies have been reported on the utility of CPs in stimulation of cancer or tumor cells in the literature. Here we report a facile fabrication method of self-doped sulfonated polyaniline (SPAN)-based interdigitated electrodes (IDEs) for controlled electrical stimulation of human osteosarcoma (HOS) cells. Increased degree of sulfonation was found to increase the SPAN conductivity, which in turn improved the cell attachment and cell growth without electrical stimulation. However, an enhanced cell growth was observed under controlled electrical (AC) stimulation at low applied voltage and frequency (≤800 mV and ≤1 kHz). The cell growth reached a maximum threshold at an applied voltage or frequency and beyond which pronounced cell death was observed. We believe that these organic electrodes may find utility in electrical stimulation of cancer or tumor cells for therapy and research and may also provide an alternative to the conventional metal-based electrodes.
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Compostos de Anilina/química , Estimulação Elétrica , Eletrodos , Osteossarcoma/fisiopatologia , Ácidos Sulfônicos/química , Fosfatase Alcalina/metabolismo , Humanos , Osteossarcoma/enzimologia , Osteossarcoma/patologia , Células Tumorais CultivadasRESUMO
The growing demand for better implant aesthetics has led to increased research on the development of all-ceramic dental implants. The use of microtextured coatings with enhanced properties has been presented as a viable way to improve tissue integrability of all-ceramic implants. The aim of this study was to evaluate the effects of different densities of anisotropic microtextured silica thin films, which served as a model coating, on the behavior of human osteoblast-like cells. The differential responses of human osteoblast-like cells to anisotropic silica microtextures with varying densities, produced via a combination of sol-gel and soft lithography processing, were evaluated in terms of alignment, elongation (using fluorescence microscopy), overall cellular activity, and the expression/activity levels of alkaline phosphatase (ALP). Statistical analysis was conducted using one-way ANOVA/Tukey HSD post hoc test. The thin films were thoroughly characterized via scanning electron microscopy/energy dispersive spectroscopy, Fourier transform infrared, and contact angle measurements. Thin film characterization revealed increased nanoscale roughness and reduced wettability on the micropatterned surfaces. Cell culture experiments indicated that the microtextures induced cell alignment, elongation, and guided colonization on the surface. Cells cultured on denser micropatterns exhibited increased metabolic activity (t = 14-21 days). The early expression/activity levels of ALP released into the medium were found to be significantly higher only on the least dense micropattern. These results suggest the possibility that microstructured silica thin films could be used to guide and enhance peri-implant cell/tissue responses, potentially improving tissue integration for metallic and all-ceramic dental implants.
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Regeneração Tecidual Guiada Periodontal/métodos , Dióxido de Silício/química , Fosfatase Alcalina/metabolismo , Regeneração Óssea , Substitutos Ósseos/química , Linhagem Celular , Proliferação de Células , Forma Celular , Cerâmica/química , Materiais Revestidos Biocompatíveis/química , Implantes Dentários , Materiais Dentários/química , Humanos , Teste de Materiais , Osteoblastos/citologia , Osteoblastos/fisiologia , Propriedades de Superfície , MolhabilidadeRESUMO
Multiple gene transfections are often required to control the differentiation of embryonic stem cells. This is typically done by removing the cells from the culture substrate and conducting gene transfection via bulk electroporation (in suspension), which is then followed by further culture. Such repetitive processes could affect the growth and behavior of delicate/scarce adherent cells. We have developed a novel nanofiber-based sandwich electroporation device capable of in situ and in culture gene transfection. Electrospinning was used to deposit poly(ε-caprolactone)/gelatin nanofibers on the Al(2)O(3) nanoporous support membrane, on top of which a polystyrene microspacer was thermally bonded to control embryonic stem cell colony formation. The applicability of this system was demonstrated by culturing and transfecting mouse embryonic stem cells. Measurements of secreted alkaline phosphatase protein and metabolic activity showed higher transfection efficacy and cell viability compared to the conventional bulk electroporation approach.
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Eletroporação/métodos , Células-Tronco Embrionárias/fisiologia , Técnicas de Transferência de Genes , Nanofibras/química , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Camundongos , Nanofibras/administração & dosagemRESUMO
Guided cell migration plays a crucial role in tumor metastasis, which is considered to be the major cause of death in cancer patients. Such behavior is regulated in part by micro/nanoscale topographical cues present in the parenchyma or stroma in the form of fiber-like and/or conduit-like structures (e.g., white matter tracts, blood/lymphatic vessels, subpial and subperitoneal spaces). In this paper we used soft lithography micromolding to develop a tissue culture polystyrene platform with a microscale surface pattern that was able to induce guided cell motility along/through fiber-/conduit-like structures. The migratory behaviors of primary (glioma) and metastatic (lung and colon) tumors excised from the brain were monitored via time-lapse microscopy at the single cell level. All the tumor cells exhibited axially persistent cell migration, with percentages of unidirectionally motile cells of 84.0 ± 3.5%, 58.3 ± 6.8% and 69.4 ± 5.4% for the glioma, lung, and colon tumor cells, respectively. Lung tumor cells showed the highest migratory velocities (41.8 ± 4.6 µm h(-1)) compared to glioma (24.0 ± 1.8 µm h(-1)) and colon (26.7 ± 2.8 µm h(-1)) tumor cells. This platform could potentially be used in conjunction with other biological assays to probe the mechanisms underlying the metastatic phenotype under guided cell migration conditions, and possibly by itself as an indicator of the effectiveness of treatments that target specific tumor cell motility behaviors.
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Neoplasias/patologia , Imagem com Lapso de Tempo/métodos , Movimento Celular , Humanos , Neoplasias/metabolismo , Poliestirenos/química , Propriedades de Superfície , Imagem com Lapso de Tempo/instrumentaçãoRESUMO
Insulin-expressing islet-like cell clusters derived from precursor cells have significant potential in the treatment of type-I diabetes. Given that cluster size and uniformity are known to influence islet cell behavior, the ability to effectively control these parameters could find applications in the development of anti-diabetic therapies. In this work, we combined micro and nanofabrication techniques to build a biodegradable platform capable of supporting the formation of islet-like structures from pancreatic precursors. Soft lithography and electrospinning were used to create arrays of microwells (150-500 µm diameter) structurally interfaced with a porous sheet of micro/nanoscale polyblend fibers (~0.5-10 µm in cross-sectional size), upon which human pancreatic ductal epithelial cells anchored and assembled into insulin-expressing 3D clusters. The microwells effectively regulated the spatial distribution of the cells on the platform, as well as cluster size, shape and homogeneity. Average cluster cross-sectional area (~14000-17500 µm(2)) varied in proportion to the microwell dimensions, and mean circularity values remained above 0.7 for all microwell sizes. In comparison, clustering on control surfaces (fibers without microwells or tissue culture plastic) resulted in irregularly shaped/sized cell aggregates. Immunoreactivity for insulin, C-peptide and glucagon was detected on both the platform and control surfaces; however, intracellular levels of C-peptide/cell were ~60 % higher on the platform.
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Técnicas de Cultura de Células/instrumentação , Regulação da Expressão Gênica , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Microtecnologia/instrumentação , Nanotecnologia/instrumentação , HumanosRESUMO
Modified Portland cement porous scaffolds with suitable characteristics for load-bearing bone tissue engineering applications were manufactured by combining the particulate leaching and foaming methods. Non-crosslinked polydimethylsiloxane was evaluated as a potential reinforcing material. The scaffolds presented average porosities between 70 and 80% with mean pore sizes ranging from 300 µm up to 5.0 mm. Non-reinforced scaffolds presented compressive strengths and elastic modulus values of 2.6 and 245 MPa, respectively, whereas reinforced scaffolds exhibited 4.2 and 443 MPa, respectively, an increase of â¼62 and 80%. Portland cement scaffolds supported human osteoblast-like cell adhesion, spreading, and propagation (t = 1-28 days). Cell metabolism and alkaline phosphatase activity were found to be enhanced at longer culture intervals (t ≥ 14 days). These results suggest the possibility of obtaining strong and biocompatible scaffolds for bone repair applications from inexpensive, yet technologically advanced materials such as Portland cement.